Our study focused on the development of non-covalent proteasome inhibitors. Most target compounds were more potent than the approved drug carfilzomib. All the tested compounds exhibited potent anti-proliferative activities. Compound 35 displayed potent ex vivo and in vivo proteasome inhibitory activities. The t1/2 of compound 35 was 2-fold longer than the analogue without piperidine ring.